1. Field of the Invention
The present invention is directed to a method of patterning materials, such as proteins, on a contoured surface by depositing them onto protrusions on the surface and to a cell containment device that may be constructed by this method.
2. Description of the Related Art
A number of methods in biochemistry require a technique for patterning single cells with a high degree of spatial selectivity. Many laboratories are trying to take advantage of the high sensitivity of living cells in sensing units for biosensor applications; in these systems, the cells must be located precisely on the circuitry of the device. In tissue engineering, it may be useful to pattern different kinds of cells on different areas of a substrate in order to build defined architecture into multifunctional tissues. Automated technologies for high-throughput screening require the placement of cells in well-ordered arrays that can be addressed individually. Basic studies of cellular function and metabolism will also benefit from the ability to control the microenvironment of patterned cells, and to perturb them individually. Assays aimed at identifying the phenotype of a cell in a population of heterogeneously transfected cells might be simplified if individual cells were localized; the production of a fluorescently labeled gene product could be detected at the location, rather than remaining unidentified in solution.
Although cell-based assays are commonly available, applications involving patterned single cells have been limited by technological problems: e.g., the selective delivery of small volumes of liquid to a well with a 50 μm diameter, the placement of cells on a defined grid, and the prevention of non-selective adhesion and cell migration. Improving the technology to generate regular arrays of cells would make it possible to develop: i) analytical systems based on single cells for the detection of toxic agents; ii) systems for high-throughput screening of combinatorial libraries and gene products; iii) research tools to study the effect of the adhesive environment on the behavior of a cell; iv) new methods for the study of cellular function and metabolism at the level of single cells and individually isolated groups of cells.
To pattern single mammalian cells onto a substrate, the best strategy is often to pattern adhesive extracellular matrix (ECM) proteins onto that substrate. Current methods for patterning ECM proteins use self-assembled monolayers (SAMs) of alkanethiolates on gold, or of alkyltrichlorosiloxanes on silicon. Previously, it has been shown that mammalian cells can adhere to flat or contoured gold surfaces patterned with SAMs. Earlier work on patterning cells focused on their interaction with SAMs of alkylsiloxanes. Photolithographic patterning of siloxanes allowed the definition of patterns of functional groups that were recognized nonspecifically by various types of cells; complex biological ligands, however, are not compatible with these photolithographic methods. Microcontact printing has also been used to directly print patterns of proteins onto surfaces. (See U.S. Pat. Nos. 5,776,748 and 5,976,826, which are hereby incorporated by reference in their entirety.)
You et al. coated arrays of large wells (1-mm diameter) non-selectively with ECM proteins; mink lung cells were forced into the wells from a suspension by dragging a flat piece of PDMS across the array of wells. (You, A. J.; Jackman, R. J.; Whitesides, G. M.; Schreiber, S. L. Chem. & Biol. 1997, 4, 969-975.) Parce et al. used gravitational sedimentation to deposit cells in arrays of 50 μm wells that were fabricated using silicon micromachining. (Parce, J. W.; Owicki, J. C.; Kercso, K. M.; Sigal, G. B.; Wada, H. G.; Muir, V. C.; Bousse, L. J.; Ross, K. L.; Sikic, B. I.; McConnell, H. M. Science 1989, 246, 243-247.) Methods have also been developed for patterning cells onto surfaces using elastomeric membranes as resists against the adsorption of proteins and the adhesion of cells. (Ostuni, E; Kane, R.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir, 2000, 16, 7811-7819.)